Department of Plant Pathology, Kansas State University, Manhattan 66506.
United States Department of Agriculture-Agricultural Research Service, Foreign Disease-Weed Science Research Unit, Fort Detrick, MD 21702.
Plant Dis. 2018 Dec;102(12):2550-2559. doi: 10.1094/PDIS-03-18-0512-RE. Epub 2018 Oct 15.
Wheat blast, caused by the Magnaporthe oryzae Triticum pathotype, is an economically important fungal disease of wheat. Wheat blast symptoms are similar to Fusarium head scab and can cause confusion in the field. Currently, no in-field diagnostic exists for M. oryzae Triticum. Loop-mediated isothermal amplification (LAMP) primers were designed to target the PoT2 and MoT3 loci, previously shown to be specific for M. oryzae and M. oryzae Triticum, respectively. Specificity was determined using 158 M. oryzae strains collected from infected wheat and other grasses and representing geographic and temporal variation. Negative controls included 50 Fusarium spp. isolates. Sensitivity was assessed using 10-fold serial dilutions of M. oryzae Triticum gDNA. PoT2- and MoT3-based assays showed high specificity for M. oryzae and M. oryzae Triticum, respectively, and sensitivity to approximately 5 pg of DNA per reaction. PoT2 and MoT3 assays were tested on M. oryzae Triticum-infected wheat seed and spikes and identified M. oryzae and M. oryzae Triticum, respectively, using a field DNA extraction kit and the portable Genie II system. The mitochondrial NADH-dehydrogenase (nad5) gene, an internal control for plant DNA, was multiplexed with PoT2 and MoT3 and showed results comparable with individual assays. These results show applicability for M. oryzae Triticum field surveillance, as well as identifying nonwheat species that may serve as a reservoir or source of inoculum for nearby wheat fields.
由稻瘟病菌（Magnaporthe oryzae）小麦专化型引起的小麦穗疫病是一种对小麦具有重要经济意义的真菌病害。小麦穗疫病的症状与镰刀菌顶腐病相似，可能会在田间造成混淆。目前，尚无针对稻瘟病菌小麦专化型的田间诊断方法。环介导等温扩增（LAMP）引物是针对先前被证明分别特异性针对稻瘟病菌和稻瘟病菌小麦专化型的 PoT2 和 MoT3 基因座设计的。使用从感染小麦和其他禾本科植物中收集的 158 株稻瘟病菌株，以及代表地理和时间变化的菌株，确定了特异性。阴性对照包括 50 株镰刀菌属分离株。使用稻瘟病菌小麦专化型 gDNA 的 10 倍系列稀释液评估了敏感性。基于 PoT2 和 MoT3 的检测方法分别对稻瘟病菌和稻瘟病菌小麦专化型具有高度特异性，对每个反应的 DNA 灵敏度约为 5 pg。PoT2 和 MoT3 检测方法用于感染稻瘟病菌小麦的种子和穗，使用现场 DNA 提取试剂盒和便携式 Genie II 系统分别鉴定出稻瘟病菌和稻瘟病菌小麦专化型。将线粒体 NADH 脱氢酶（nad5）基因作为植物 DNA 的内参与 PoT2 和 MoT3 进行多重扩增，结果与单独的检测方法相当。这些结果表明，该方法适用于稻瘟病菌小麦专化型的田间监测，以及鉴定可能作为附近小麦田接种体来源或储存库的非小麦物种。
